Perirhinal Cortex Lesions Produce Retrograde Amnesia for Spatial Information in Rats: Consolidation Or Retrieval? Juan M.J

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Research Perirhinal cortex lesions produce retrograde amnesia for spatial information in rats: Consolidation or retrieval? Juan M.J. Ramos1 Departamento de Psicología Experimental y Fisiología del Comportamiento, Facultad de Psicología, Campus de Cartuja, Universidad de Granada, Granada 18071, Spain

Several lines of evidence in humans and experimental animals suggest that the hippocampus is critical for the formation and retrieval of spatial memory. However, although the hippocampus is reciprocally connected to adjacent cortices within the medial temporal lobe and they, in turn, are connected to the neocortex, little is known regarding the function of these cortices in memory. Here, using a reference spatial memory task in the radial maze, we show that neurotoxic perirhinal cortex lesions produce a profound retrograde amnesia when learning–surgery intervals of 1 or 50 d are used (Experiment 1). With the aim of dissociating between consolidation and retrieval processes, we injected lidocaine either daily after training (Experiment 2) or before a retention test once the learning had been completed (Experiment 3). Results show that reversible perirhinal inactivation impairs retrieval but not consolidation. However, the same procedure followed in Experiment 2 disrupted consolidation when the lidocaine was injected into the dorsal hippocampus. The results of Experiment 4 rule out the possibility that the deficit in retrieval is due to a state-dependent effect. These findings demonstrate the differential contribution of various regions of the medial temporal lobe to memory, suggesting that the perirhinal cortex plays a key role in the retrieval of spatial information for a long period of time.

Since the discovery of the dramatic retrograde amnesia induced network of structures (Ross and Eichenbaum 2006; Texeira et al. by bilateral damage to the medial temporal lobe in the patient 2006; Ji and Wilson 2007). However, the anatomical routes by HM, the hippocampus and its related cortices (entorhinal, peri- which the hippocampal region accesses the neocortical represen- rhinal, and postrhinal) have been the subject of a great number tation of episodes or facts are not known. In terms of connectiv- of studies about the brain systems involved in long-term memory ity, the PRC is ideally located to link the hippocampus with as- formation and retrieval (Milner et al. 1998; Squire et al. 2004; sociation cortices involved in different aspects of spatial cogni- Frankland and Bontempi 2005). Over the last 15 years, many tion (Scharfman et al. 2000). Supporting this idea, several studies clinical observations in humans, for whom detailed neuropath- have shown that prefrontal cortex or amygdala inputs can facili- ological and neuropsychological information is available, have tate the transfer of hippocampal activity to the neocortex via an revealed that after limited lesions to the CA1 field of the hippo- enhancement of entorhinal to perirhinal communication, so it campus, retrograde amnesia is temporally graded, covering only may be that the perirhinal cortex is a necessary interface between a few years (Zola-Morgan et al. 1986; Manns et al. 2003). Inter- the hippocampus and a distributed neocortical network for long- estingly, when the damage occurs in the hippocampus plus the term memory (Paz et al. 2006, 2007). In addition, functional adjacent cortices, some studies have found a more profound and studies in which the PRC is hyperactivated by means of excito- extended retrograde amnesia, sometimes lasting decades (Reed toxic or electrolytic lesions have demonstrated increased c-Fos and Squire 1998; Bayley et al. 2006; for review, see Squire and expression in the parietal, retrosplenial, and frontal cortices, re- Bayley 2007), while others have shown an ungraded memory loss gions that are all involved in the long-term representation of (Moscovitch et al. 2006). The differential magnitude and exten- spatial memory (Glenn et al. 2005; see also Maviel et al. 2004). sion of retrograde amnesia following limited hippocampal dam- Several studies have examined, with different behavioral age versus large medial temporal lobe lesions suggest that the paradigms and species, the effect of PRC lesions on memory con- adjacent cortices have an essential function in memory forma- solidation or retrieval. When object discrimination problems and tion or retrieval. Thus, although several lines of research indicate permanent lesions have been used, results have been inconsis- that the hippocampus is necessary for the initial retrieval and tent. Some studies have found a profound graded retrograde am- consolidation of declarative memory (for review, see Frankland nesia (Wiig et al. 1996; Kornecook et al. 1999) and others only a and Bontempi 2005), the specific contribution of the different mild, transient amnesia (Mumby and Glenn 2000). In monkeys, cortices in the medial temporal lobe to the formation of long- rhinal cortex removal produced retrograde amnesia for two sets term memory or retrieval is not yet well understood. of object discrimination problems (each set having 60 problems) The present study focuses on the perirhinal cortex (PRC), learned 16 wk or 1 wk before surgery; however, the lesions did and it uses a spatial paradigm in rats. It has been proposed that not disrupt postoperative acquisition and retention of similar the encoding, retrieval, and consolidation of declarative memory problems, suggesting that this cortex is critical for consolidation rely on interactions between the hippocampus and a cortical and/or retrieval of object discrimination problems (Thornton et al. 1997). Other investigations, using reversible lesions, have demonstrated the involvement of the PRC in encoding, retrieval, 1Corresponding author. and consolidation stages in object-recognition tasks (Winters E-mail [email protected]; fax 34-958-246239. and Bussey 2005a,b). In relation to contextual memory, Burwell Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.1036308. et al. (2004) showed clearly that the PRC is involved in the stor-

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Retrograde amnesia after perirhinal cortex lesion

age and/or retrieval of the memory for contextual fear for at least Histology 100 d after learning. In contrast with the foregoing, however, Tissue damage was microscopically identified by marked thin- there has been no observation of a consistent retrograde amnesia ning of the cortex, necrosis, or missing tissue. No important dif- in tasks using complex contextual information, particularly in ferences were found between the histological results of the four allocentric tasks, where subjects must recall spatial relations be- lesioned groups used in this experiment: perirhinal cortex le- tween landmarks in order to locate a goal. In these studies only a sions/relearning 1 d (PRC-rl 1 d), perirhinal cortex lesions/ slight and transient retrograde amnesia in early probe trials has relearning 50 d (PRC-rl 50 d), perirhinal cortex lesions/learning 1 been observed in the Morris water maze (Mumby and Glenn d (PRC-l 1 d), and perirhinal cortex lesions/learning 50 d (PRC-l 2000; Glenn et al. 2003). 50 d). The lesions, aimed at areas 35 and 36, were generally lim- One problem with the above studies is that lesions made to ited to the target area, creating a longitudinal groove on both the PRC before training cause a profound anterograde amnesia in sides of the rhinal fissure (Fig. 1). In general, the lesion affected object-recognition memory and contextual fear tasks (Bucci et al. the six layers of the PRC and did not reach (except in two animals 2000; Gaffan et al. 2000; Murray et al. 2007). Thus, with these belonging to group PRC-l 50 d) the postrhinal cortex. In four paradigms, when the lesions are made after the training and ret- cases, the cellular damage affected partially the CA1 field bilat- rograde amnesia is observed, it is difficult to determine if the erally in the ventral hippocampus. In 20% of the animals, the lesions have affected the performance of the task or a process of lateral entorhinal cortex was minimally affected to varying de- consolidation and/or retrieval. In relation to spatial memory, al- grees. When the lateral entorhinal cortex was partially lesioned though there is debate regarding the function of the PRC in ac- (from 3% to 17%), the damaged area was intermediate, between quisition (Muir and Bilkey 2001; Aggleton et al. 2004), several 4 and 5.5 mm posterior to bregma according to the Paxinos and studies have shown that rats with PRC lesions have intact acqui- Watson (1998) atlas. Apparently, in no case (except in the two sition in a radial maze (Bussey et al. 1999; see also Winters et al. aforementioned subjects with partial postrhinal lesions) did the 2004 in a Y-maze). For this reason, the present study uses a spatial most caudal portion of the PRC suffer lesions, and the dorsolat- reference memory test in a four-arm plus-shaped maze, for which eral band of the entorhinal cortex was spared in all cases. In the our laboratory has previously shown the absence of deficits in latter region, spatially modulated neurons have been described acquisition/performance following perirhinal lesions (Ramos recently, and lesion studies have shown its involvement in spa- 2002). In sum, using a hippocampal-dependent spatial reference memory paradigm (Ramos 2000, see Experiment 1), the principal aim of the present study was to test the idea that the PRC influences spatial memory formation or retrieval. Results Experiment 1: Neurotoxic lesions of the perirhinal cortex 1 or 50 d after learning The aim of this experiment was to investigate whether damage to the PRC produces retrograde amnesia in rats that had learned a reference spatial memory test presurgically. A four-arm plus- shaped maze was used in such a way that three arms were for starting and the fourth was the goal arm. Presurgically, rats received eight trials per session and one session per day to reach a learning criterion (14 correct trials on two consecutive days). After either a 24-h period (1 d groups) or 49–50 d (50 d groups) following the last training session, rats received bilateral N- methyl-D-aspartate (NMDA) or sham lesions in the PRC. Eight days later, we examined spatial performance in various ways. First, all the rats received retraining on the same spatial task for 12 consecutive days. The assumption was that preoperative acquired spatial memory would facilitate relearning. In addition, separate groups of rats with perirhinal or control lesions, with no prior training, were used to assess the effects of previous training on any new learning during the postsurgery performance. Better performance during the retraining phase in the control- relearning group, as compared to the control-learning group, would reflect memory for the original place. An important ob- jective of this experiment was to investigate whether this holds true for rats with perirhinal lesions. Second, on the first day of retraining, no reward was present in the food cup of the goal arm, and the average number of trials needed to reach three correct trials was determined for each rat. The rationale was that a greater preoperative memory would be reflected in more accurate Figure 1. (A) Lateral view of a representative perirhinal lesion and pho- spatial behavior during the first day of postoperative testing. tomicrographs of coronal sections stained with cresyl violet from a rep- Thus, since no reward was present on this first day of retraining, resentative lesioned rat. (B) Coronal sections showing the (gray) largest and (central white area) smallest perirhinal lesions in the lesioned groups the performance of the rats represents a relatively pure indication of Experiment 1. No important differences were observed between the of preoperative spatial memory, not contaminated by learning lesioned groups. In A and B, anteroposterior coordinates are according to effects. bregma (Paxinos and Watson 1998). www.learnmem.org 588 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

tial memory (Fyhn et al. 2004; Steffenach et al. 2005). Finally, some animals showed minor damage (from 2% to 20%) in the cortex slightly dorsal to the PRC, the ventral temporal association cortex. In order to quantify the extension of the lesion in each lesioned rat, regions of cell loss and gliosis identified microscopically were plotted on drawings of coronal sections from the atlas of Paxinos and Watson (1998). For each perirhinal-lesioned rat, the reconstruction of the lesion was made based on eight coronal sections (anteroposterior levels: 3.3, 3.8, 4.3, 4.8, 5.2, 5.6, 6.0, and 6.3 mm posterior to bregma according to Paxinos and Wat- son 1998). Each coronal section was digitized, and the lesioned area was measured in square millimeters by a computer program (Autocad, version 2004). The anatomical limits of the perirhinal, SEM) number of errors committed until reachingע) entorhinal and postrhinal cortices were defined using the works Figure 2. Mean by Burwell and associates (Burwell et al. 1995; Burwell and Ama- criterion during the retraining phase of Experiment 1. ral 1998). The volume of damage was expressed as a percentage of normal volume obtained from three normal non-lesioned rats. control rats in the retraining phase of this experiment. Retraining –learning ן Table 1 represents the mean percentage of damage following began 8 d after surgery. A two-way ANOVA (lesion the PRC lesions. It can be seen that area 35 was slightly more surgery interval) indicated a significant effect of lesion (F1,44 le- affected than area 36. Also, when the lateral entorhinal cortex sion = 48.9, p < 0.0001) and of learning–surgery interval (F1,44 was affected, the extension of the lesion was less than when the learning–surgery interval = 5.73, p < 0.02) but not of interaction ventral temporal association cortex was lesioned unintention- (F1,44 interaction = 1.50, p = 0.22). ally, and there was thus a tendency for the lesion to be diverted It is interesting to point out that, considering only the experimental groups, the mean number of errors to criterion was ,0.39מ = dorsally. Importantly, neither in the PRC-rl1d(r -p = 0.5) was a signif- similar in the PRC-rl 1-d and the PRC-rl 50-d groups, no signifi ,0.19מ = p = 0.14) nor in the PRC-rl 50 d (r icant correlation observed between the size of PRC lesion (area cant differences between them being detected (p = 0.41). How- 35 + area 36) and performance during the relearning phase. Fur- ever, if only the two control groups are considered, significant thermore, the performance of the perirhinal-lesioned rats was differences do appear, showing, as expected, significantly more not related to the amount of accompanying damage in adjacent forgetting by the Control-rl 50-d group than by the Control-rl 1-d structures. During relearning, no significant correlation was de- group (p < 0.01). tected between the size of the entorhinal cortex lesion and per- During the first day of retraining, no reward was placed in formance (r = 0.22, p = 0.42), the size of the ventral temporal the food cup of the goal arm, and therefore performance on that association cortex lesion and performance (r = 0.18, p = 0.53) or day represents a relatively pure measure of retention not con- the entorhinal plus ventral temporal association cortex and per- taminated by learning processes. In order to make the memory of formance (r = 0.24, p = 0.4). the preoperative information not depend on a single probe trial, we determined for each rat the number of trials necessary on this Behavioral results first day to reach three correct trials. A previous pilot experiment performed in our lab showed that during the reversal learning of Preoperative learning the task used in the present study, in which the goal arm was the During the learning phase, the four main groups of subjects ac- one opposite to the arm used during the learning phase, normal quired the spatial reference memory task at the same rate. Thus, rats consistently looked for the original goal for an average of 5.9 a two-way ANOVA with two between-group variables (lesion/no trials. Extinction generally appeared in the final part of a training lesion and 1/50 d) did not detect significant differences in session consisting of eight trials, thus enabling the animal’s relation to the number of incorrect trials effected before reach- memory to be evaluated for a longer period of time. This proce- ing criterion (PRC-rl 1-d = 35.1, Control-rl 1-d = 32.1, PRC-rl dure has the further advantage of allowing the first trials to serve F p 50-d = 34.0, Control-rl 50-d = 32.5; 1,44 lesion = 2.34, = 0.13; as reminders, facilitating the memory of the previously learned F p F 1,44 learning–surgery interval = 0.59, = 0.59; 1,44 interac- material without incurring in any relearning. Based on this ration = 0.08, p = 0.76). tionale, Figure 3 depicts the mean number of trials needed to reach three correct trials on the first postoperative day of testing. learning–surgery interval) revealed ן Postoperative retention A two-way ANOVA (lesion Figure 2 depicts the mean number of errors committed until cri- that the lesioned groups needed a significantly greater number of terion is reached again (or, in its absence, the mean number of trials than the control rats to reach three correct trials (F1,44 le- errors during 12 consecutive days) by perirhinal-lesioned and sion = 11.48, p < 0.001). As expected, the 50-d groups showed

Table 1. Percent damage following PRC lesion Group n Area 36 Area 35 LE ME POC TeA

0.7 ע 6.5 0 0 1.1 ע 3.4 7.8 ע 52.5 4.1 ע PRC-rl (1 d) 13 46.9 1.6 ע 8.0 0 0 0.8 ע 4.1 6.5 ע 54.0 3.1 ע PRC-rl (50 d) 12 40.9 1.1 ע 5.1 0 0 0.8 ע 4.2 7.4 ע 60.8 4.6 ע PRC-l (1 d) 7 54.0 1.3 ע 5.6 1.6 ע 2.0 1.4 ע 1.6 0.6 ע 5.0 8.1 ע 47.5 4.2 ע PRC-l (50 d) 10 48.9

.SEM. The lesions were intended to encompass both areas 35 and 36 ע Data represent mean ME, medial entorhinal cortex; LE, lateral entorhinal cortex; PRC-rl, perirhinal cortex relearning; PRC-l, perirhinal cortex learning; POC, postrhinal cortex; TeA, ventral temporal association cortex. www.learnmem.org 589 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

case, no significant differences were observed between the PRC-rl group and the Control-rl group (p = 0.35). However, a profound deficit for the PRC-rl group was evident when the performance of this group during the 12 d of retraining was compared to that of the Control-rl group (p < 0.0006). Furthermore, as expected, the performance of the Control-rl group was significantly better than that of the two groups without previous learning, showing that the Control-rl rats remembered well the location of the goal learned presurgically (Control-rl vs. Control-l, p < 0.0003; Con- trol-rl vs. PRC-l, p < 0.0003). Other Newman-Keuls comparisons indicated no significant differences during relearning between the PRC-rl and the PRC-l groups (p = 0.69) and between the PRC-rl and the Control-l groups (p = 0.54). In addition, a two- days) comparing the PRC-rl, PRC-l, and ן way ANOVA (group SEM) number of trials needed to reach three correct Control-l groups during relearning revealed that the performanceע) Figure 3. Mean trials on the first postoperative day of testing of Experiment 1 (without of the three groups was similar, no significant differences being reward). found among groups (F2,24 groups = 0.49, p = 0.61) or in the interaction (F22,264 interaction = 0.56, p = 0.94), although for more forgetting than the 1-d groups, the former needing signifi- the days factor, significant differences were found (F11,264 cantly more trials on the first day of testing to be able to perform days = 15.15, p < 0.0001). three correct trials (F1,44 learning–surgery interval = 5.17, ן p < 0.02). The interaction between lesion learning–surgery in- Experiment 2: Perirhinal cortex lidocaine injection terval was not found to be significant (F1,44 interaction = 0.09, p = 0.76). Also of interest was whether the differences between immediately after training session lesioned and control groups were significant in the different Experiment 1 shows that permanent neurotoxic lesions of the learning–surgery intervals under consideration. Post hoc New- PRC produce a profound impairment in the expression of spatial man-Keuls comparisons indicated that these differences were significant for groups PRC-rl 1 d versus Control-rl1d(p < 0.03) but not significant for 50-d groups (PRC-rl 50 d vs. Control-rl 50 d, p = 0.1). In order to study any possible effect of the perirhinal lesions on the performance of the allocentric task used in the present experiment, we included four additional groups of rats during the 12 d of retraining. They were different from the four groups trained presurgically only in that they had received no previous training. Figure 4A shows the performance during the 12 consecutive days of retraining of the animals operated on 1 d after the end of learning and of their two respective additional groups (days ן without previous training. A two-way ANOVA (group revealed a significant effect of lesion (F3,36 = 16.09, p < 0.0001), days (F11,396 = 26.64, p < 0.0001), and interaction (F33,396 = 1.84, p < 0.003). Post hoc Newman-Keuls tests showed that during the first day of retraining, the Control-rl group remembered the location of the goal arm significantly better than the PRC-rl group (p < 0.02). Importantly, throughout the 12 days of retraining, the Control-rl group performed significantly better than the rest, suggesting that the spatial memory it acquired during the preoperative phase was very much superior to that of other groups (Control-rl vs. PRC-rl, p < 0.0001; Control-rl vs. PRC-l, p < 0.0002; Control-rl vs. Control-l, p < 0.0001). Additionally, no significant differences were detected upon comparing the rest of the groups to each other, which suggests that the perirhinal lesions did not worsen the performance/acquisition of the task but did impair the remembering of the preoperative information (PRC-rl vs. PRC-l, p = 0.57; PRC-rl vs. Control-l, p = 0.91; PRC-l vs. Control-l, p = 0.37). Figure 4B shows the performance during the 12 consecutive days of retraining of the animals operated on 50 d after the end of the learning and of their respective additional groups without days) indicated a ן previous training. A two-way ANOVA (group significant effect of lesion (F3,38 = 9.06, p < 0.0001) and days (F = 27.38, p < 0.0001) but not of interaction (F = 1.03, ע 33,418 11,418 p = 0.41). It was of interest to know whether there were differ- Figure 4. Mean ( SEM) percentage of correct responses observed in the perirhinal lesioned (PRC-rl trained presurgically and PRC-l without ences between the additional groups and the groups with previ- prior experience) and control groups (Control-rl and Control-l) of Experi- ous training, and for this reason the pertinent Newman-Keuls ment 1, during the 12 consecutive days of retraining. In A the learning– comparisons were made. During the first day of retraining, in this surgery interval was 1 d, and in B it was 50 d. www.learnmem.org 590 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

information learned presurgically. However, permanent lesions cannot provide certain information, such as at what stage of memory (encoding, consolidation, or retrieval) is a particular brain region necessary. The goal of Experiment 2 was to look into whether the PRC retrograde amnesia seen in Experiment 1 was caused by a disruption of a process of consolidation. Several studies have shown retrograde amnesia for spatial information following posttraining intra-hippocampal infusion of anisomycin (Rossato et al. 2006), LY326325—a selective AMPA/kainite recep- tor antagonist (Riedel et al. 1999), or lidocaine (Broadbent et al. 2006), showing that any of these procedures is suitable for producing retrograde amnesia when the neural inactivation of the hippocampus is induced just after training. Thus, in the present experiment, an intra-perirhinal infusion of lidocaine or sham infusion was administered immediately after each daily training session. This ensures that there was no interference with normal PRC function during either acquisition or retrieval phases, allowing any amnesic effect to be due only to consolidation disruption. A second experimental group that received an intra- hippocampal infusion of lidocaine immediately after each daily training session was used in the present experiment to confirm that the exact procedure followed is suitable for inducing retrograde amnesia when the target structure has a consolidation function. To accelerate the acquisition of the task as much as possible and to reduce the number of injections of lidocaine, we intro- duced in this experiment a slight modification with regard to the previous experiment. On this occasion, throughout the acquisition period, a piece of yellow sandpaper was positioned on the Figure 5. (A) Schematic representation of infusion needle tips in the floor of the goal arm. Training ended when each rat reached a dorsal hippocampus and the perirhinal cortex for each of the experimen- learning criterion of at least 14 correct trials on two consecutive tal rats used in Experiment 2. Each black dot shows the injection site for a single cannula per animal. Anteroposterior coordinates according to days. Lidocaine or buffer was injected into each rat immediately SEM) percentage of correctע) Paxinos and Watson (1998). (B) Mean after each daily training session including the day that the ani- responses of the three groups used in Experiment 2 during the transfer mal reached criterion. The day after reaching criterion, each rat test, 1 d after reaching criterion. underwent a transfer test (with eight trials) in order to assess the animal’s memory of the goal arm. Importantly, during this transfer, the sandpaper covering the goal arm was removed so that the Experiment 3: Perirhinal cortex lidocaine injection only guide available to the rats to solve the test was the configu- before memory test ration of the extramaze stimuli. We hypothesize that if the ret- The goal of this experiment was to investigate whether the PRC rograde amnesia induced by permanent neurotoxic PRC lesions is necessary for the retrieval of spatial memory. For this reason, in is due to the interruption of a process of consolidation, daily this experiment the injections of lidocaine or buffer were made post-training intra-perirhinal infusion of lidocaine would pro- just before a memory test that took place 1 d after the animals duce retrograde amnesia during the transfer test. reached the learning criterion (14 correct trials on two consecutive days). This memory test started 5 min after the injections Histology and was comprised of eight trials similar to those taking place Figure 5A depicts the tip location of the injection cannulas for during the training phase of the experiment. each of the seven perirhinal and each of the six hippocampal injected rats. All rats, including the seven sham injected subjects, Histology showed damage to the cortex overlying the injection sites due to Essentially, the tip locations of the injection cannulas were simi- the placement of the guide cannula. lar to those observed in Experiment 2, all of them being within area 36. Behavioral results During the acquisition, one-way ANOVA did not detect signifi- Behavioral results cant differences in relation to the number of errors to criterion During the acquisition, one-way ANOVA did not detect signifi- (Perirhinal = 16.3; Hippocampus = 15.7; Control = 16.6; cant differences in the number of errors to criterion (lidocaine

F2,17 = 0.37, p = 0.69). Figure 5B illustrates the performance of the injection = 33.8; buffer injection = 35.7; F1,13 = 0.08, p = 0.77). three groups during the transfer test. One-way ANOVA revealed Figure 6A illustrates the performance of the experimental and significant differences among groups (F2,17 = 12.15, p < 0.0005). control animals on the day of the lidocaine/buffer injection, and However, post hoc Newman-Keuls comparisons indicated that on the previous and following days, when no injections were -days) showed a signifi ן only the post-training intra-hippocampal lidocaine injected made. A two-way ANOVA (lidocaine group showed retrograde amnesia (Hippocampus vs. Control, cant effect of lidocaine (F1,13 = 9.03, p < 0.01), days (F2,26 = 13.17, p < 0.001; Hippocampus vs. PRC, p < 0.0006; PRC vs. Control, p < 0.0001) but not of interaction (F2,26=0.74, p = 0.48). In an p = 0.39). Therefore, the most plausible interpretation of the attempt to analyze in more depth the differences between above data is that PRC inactivation following training, in con- groups, two one-way ANOVAs of repeated measures and trend trast with hippocampal inactivation, does not interrupt the pro- analyses were performed for the experimental and control cess of consolidation. groups. For the control group, one-way ANOVA indicated no www.learnmem.org 591 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

lidocaine precisely during the retrieval process may have been provoking a state-dependent phenomenon that made the retrieval more difficult. The present experiment was a control experiment. The aim was to demonstrate that a similar inactivation using a different learning task does not induce a similar impairment in retrieval. The task used in Experiment 4 was an egocentric task. Using the same four-arm plus-shaped maze, the rats had to always turn either to the left or to the right in order to obtain the reward. The start arm varied from trial to trial. At the rate of eight trials per day, the acquisition phase ended when each rat reached a learning criterion of 14 correct trials on two consecutive days. On the following day, each rat was injected bilaterally with lidocaine in the PRC 5 min before a memory test comprised of eight trials similar to those of acquisition. Histology The location of the tips of the injection cannulas was essentially the same as observed in the previous experiments, all being limited to area 36. Behavioral results During the acquisition, one-way ANOVA did not detect significant differences in the number of errors to learning criterion

(PRC = 14.6; Control = 14.1; F1,12 = 0.17, p = 0.67). Figure 6B shows the performance of the PRC and control injected groups during the day of lidocaine/buffer injection and during the previous and following day on which no injection was made. A days) did not show significant ן two-way ANOVA (lidocaine

differences between groups (F1,12 lidocaine = 0.48, p = 0.49; F2,24 days = 2.57, p = 0.09; F2,24 interaction = 0.22, p = 0.80). In short, these results rule out the possibility that the retrieval deficit observed in Experiment 3 following PRC inactivation was due to a SEM) percentage of correct responses on the day state-dependent effect. In fact, these data reinforce the resultsע) Figure 6. Mean the lidocaine/buffer injection took place and on the previous and follow- obtained in Experiment 3 suggesting that the PRC is necessary for ing days, when no injections were made. (A) The data of Experiment 3 the retrieval of spatial memory. (allocentric task); (B) the data of Experiment 4 (non-allocentric task). Discussion significant differences during the previous/buffer/following days The main findings of the present study indicate that the PRC is necessary for the expression of spatial memory. Neurotoxic le- (F2,12 = 2.70, p = 0.08). A trend analysis showed that the linear sions in the PRC, 1 or 50 d after the end of learning, induced a component was not significant (F1,6 = 0.0008, p = 0.97), and that profound nongraded retrograde amnesia. The amnesia produced the quadratic component was marginally significant (F1,6 = 4.70, p = 0.07). For the lidocaine injected group, one-way ANOVA re- became evident both when a memory test (without reward) dur- vealed significant differences during the3dofinterest ing the first day of postoperative retention was used and also when a procedure of relearning was used. In an attempt to de- (F2,14 = 9.21, p < 0.002). Newman-Keuls tests showed a significant effect when performance on the day of the lidocaine injec- termine which process was altered by the lesions, we injected tion was compared to performance on the previous day lidocaine into the PRC immediately after each training session or (p < 0.004) and when it was compared to that of the following before a test of memory. Importantly, data showed that lidocaine day (p < 0.003). A trend analysis indicated that the quadratic inactivation after training did not impair the retention of the task when the drug was injected in the PRC but did negatively (F1,7 = 23.72, p < 0.001) but not the linear component affect retention when injected in the hippocampus, a structure (F1,7 = 0.14, p = 0.71) was significant. Finally, t-tests for indepen- dent samples were used to determine specific differences between clearly involved in the consolidation of spatial information (Rie- the control and experimental groups during each of the3dof del et al. 1999; Rossato et al. 2006). In contrast, lidocaine injected interest. Results indicated that on the day of the lidocaine/buffer into the PRC disrupted retention when the injection took place a few minutes before a spatial memory test; however, PRC inacti- injection significant differences were detected (t13 = 22.40, vation before the memory test did not affect the retention of a p < 0.04), but not on the previous (t13 = 1.19, p = 0.25) or the non-allocentric task, thus ruling out the possibility that the defi- following day (t13 = 1.37, p = 0.19). cit in retrieval of spatial memory is due to a state-dependent effect. In sum, the main data suggest that the PRC is necessary for Experiment 4: Perirhinal cortex lidocaine injection the retrieval or storage of spatial memory for a prolonged period before memory test in a non-allocentric task of time, at least 50 d, but not for consolidation. It is well known that retrieval is a context-dependent process, the It seems unlikely that the retrograde amnesia we observed in context being either external or internal. In Experiment 3, we Experiment 1 was due to a spatial navigation deficit. First, rats observed retrieval deficits when the PRC was inactivated before a with PRC lesions without previous training are able to learn the memory test; however, during the acquisition the PRC was acti- task at the same rate as the controls without learning experience, vated. So, in Experiment 3, the altered brain activity induced by suggesting a normal performance. These data agree with previous www.learnmem.org 592 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

findings showing that rats with PRC lesions, in general, do not observed amnesia is flat, not temporally graded. For example, in manifest any deficit in the acquisition of several allocentric tasks the study by Kornecook et al. (1999), which used prolonged in the radial maze (Bussey et al. 1999; Ramos 2002; Aggleton et al. learning–surgery intervals, the control group exhibited clear for- 2004; Winters et al. 2004). Second, during the 12 d of relearning, getting. Thus, the retention of the material learned 2 d prior to the perirhinal lesioned rats with preoperative training relearn the the surgery was markedly superior to that learned 58 d before task at the same rate as the control rats without previous training. surgery. However, in the perirhinal lesioned group, the amnesia Thus, our data suggest that rats with PRC damage are still able to observed for the material learned 2 d, as opposed to 58 d, prior to encode spatial cues and establish spatial relationships. These ob- surgery was identical. On this matter, our data are similar to that servations allow us to rule out the possibility that retrograde am- of Kornecook et al. (1999), despite the behavioral paradigm being nesia was caused by an alteration in perception or discrimination completely different. Likewise, a recent report on rats showed a abilities per se, an additional function that some authors have flat retrograde amnesia during a prolonged period of time (up to recently proposed for the PRC (Gaffan et al. 2000; Norman and 100 d after learning) for contextual information (Burwell et al. Eacott 2004; Murray et al. 2007). Instead, the impairment seems 2004). In consonance with the aforementioned studies involving to reflect a true loss of information. rats, other studies have found a prolonged retrograde amnesia in The simplest interpretation of the spared spatial learning in monkeys that learned object-discrimination problems 4 mo be- rats with perirhinal lesions is that the visuospatial information fore rhinal cortex ablation (Thornton et al. 1997). These studies, that accesses the hippocampus in order to solve allocentric tasks although they use paradigms different from the ones used in our does so using various routes. Among these routes, some possibili- experiments, agree with the data obtained in the present study, ties to be considered would be direct connections from the pre- suggesting that the PRC is necessary during a long period of time subiculum and parasubiculum to the fascia dentate (Witter et al. for retrieval/storage or consolidation of the information learned 1988), from the postrhinal cortex to the subiculum (Naber et al. presurgically. 2001) and from subcortical regions such as the midline nuclei of When a spatial paradigm is used, results are less clear. In two the thalamus to the hippocampus and subiculum (Wouterlood et studies carried out in the water maze using electrolytic or aspi- al. 1990; Dolleman-Van del Weel and Witter 1996). These inputs ration lesions, rats with PRC damage displayed, in general, a defi- to the hippocampus and their reciprocal outputs, in the absence cit in the early retention trials of place problems learned2dor4 of the perirhinal afferents, may be sufficient for the acquisition of wk before surgery (Mumby and Glenn 2000; Glenn et al. 2003). spatial memories (Aggleton et al. 2000). However, the central However, the deficits were small and animals recovered quickly. finding of the present study is that although perirhinal lesioned It may be that differences in procedure (type of lesion, task used, animals are able to learn a place task as well as controls, they are cues or experimental design) are contributing decisively to the profoundly incapacitated when it comes to the expression of this differences between these studies and the data obtained in the information. Therefore, after learning, hippocampal–perirhinal present study. For example, the authors themselves have shown modulation may be essential to express the recently acquired that differences as subtle as the experimental design (between- information. subjects vs. within-subjects design) or the type of lesion (electro- While this study clearly shows a necessary role for the PRC lytic vs. aspiration) contribute considerably to the development in the expression of spatial memory, previous studies in our lab of retrograde amnesia following PRC lesions (Mumby and Glenn with the same spatial paradigm showed that this cortex is not 2000; Glenn et al. 2003). Also using the Morris water maze, one necessary for retention when the PRC lesion is made before the recent study found no retrograde amnesia following PRC lesions training. Specifically, rats with PRC neurotoxic lesions retained a (Steffenach et al. 2005). Importantly, although in that study no place task 24 d after the end of learning as well as control rats did significant differences were detected between perirhinal and con- (Ramos 2002). These results agree with other studies using the trol rats, 7 d after PRC surgery, the lesioned animals remembered water maze in which rats with PRC lesions learned a place prob- the location of the platform 25% worse than the controls (see Fig. lem at a normal rate and performed as well as control rats on a 5 in Steffenach et al. 2005), indicating a tendency toward signifi- retention test 3 wk later (Mumby and Glenn 2000). One inter- cance. Comparing the results of our study with those of Steff- pretation of this pattern of results would suggest that spatial en- enach et al. (2005), we must keep in mind that the effect on coding in the intact animal engages a distributed network of retention may depend on the exact requirements of the behav- structures, one of them being the PRC (Muir and Bilkey 2001). ioral task and the specific procedure used. For example, in our After learning, this network must be activated, either in its en- study, rats received eight postoperative retention trials, without tirety or partially, in order for successful retrieval of the recently reward, the first day of postoperative testing, thus making it pos- acquired information to take place. This would explain why peri- sible to assess memory in various trials. One positive effect of this rhinal lesioned rats have serious difficulties in expressing preop- procedure is that the first few trials can have a reminding func- erative acquired information. However, when the acquisition of tion. However, in the study of Steffenach et al. (2005), the ani- spatial tasks takes place in animals with PRC lesions, the rest of mals only receive a probe trial 7 d after perirhinal surgery, and the structures involved could compensate for the function of the thus any possible reminding effect is absent. PRC, the latter not being necessary in the acquisition or in the Unlike the aforementioned studies, this study makes the subsequent expression of the information. Thus, the PRC is dif- important contribution of dissociating between different ferentially involved in spatial information retrieval, depending memory processes by means of reversible inactivation of the PRC on whether the original learning took place in an intact brain at different stages of memory. Thus, lidocaine inactivation of the (lesions made after training) or whether it occurred in a perirhi- PRC impairs the retrieval but not the formation of memory. To nal damaged brain (lesions made before training). our knowledge, this study is the first to comprehensively exam- In agreement with our results, previous research has shown ine the role of the PRC in the retrieval and consolidation of a significant implication of the PRC in the retrieval/storage or spatial memory. consolidation of information. However, important differences A point worth discussing is the type of task used in this emerge according to the behavioral paradigm used. In object- study. It has been suggested that to solve a reference memory task discrimination tasks, several studies have demonstrated retro- like the one used in our experiments, or similar to it, the animals grade amnesia (Wiig et al. 1996; Kornecook et al. 1999; Mumby use an allocentric-based strategy (Ramos 2000; Maviel et al. 2004; and Glenn 2000). In general, a detailed analysis shows that the Frankland and Bontempi 2005). In fact, in our series, this possi- www.learnmem.org 593 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

bility is not directly addressed, and therefore it cannot be ruled retrograde amnesia occurs in cases in which the damage affects out that the rats, under the environmental configuration of our adjacent cortices, such as the entorhinal and the perirhinal cor- lab, adopt a cue-based strategy. However, previous experiments tices, and not only the hippocampus (Zola-Morgan et al. 1986; performed in our lab, using the same task and apparatus in the Bayley et al. 2006). According to our data, the perirhinal cortex is same experimental room and context as in the present study, necessary for retrieval during a long period of time. It could thus suggest that the present task is most likely solved using a place- be argued that in human patients a lesion that affects the hip- based strategy, which is hippocampal-dependent, and not a cue- pocampus plus the adjacent cortices (e.g., PRC) is producing a based strategy. First, in an anterograde sense, our hippocampal double deficit: a deficit in consolidation due to hippocampal lesioned rats were profoundly impaired in their performance of damage and a deficit in retrieval due to rhinal damage, which this task, but hippocampal damage does not affect the acquisi- would give rise to a more profound amnesia. tion of a spatial cue-based problem (Ramos 2000, Experiments 1 To conclude, the formation and retrieval of spatial memory and 3). Second, in a retrograde sense, using the present task and depend on a system of structures in which the medial temporal context, hippocampal lesions induce retrograde amnesia (Ramos lobe plays a key role. However, individual structures within this 1998, Experiment 1); however, when rats learn presurgically to region make different contributions to memory processes. Here approach a single intramaze cue or a single prominent extramaze we provide new evidence that sheds light on the importance of cue, which change as the goal arm changes from trial to trial, the perirhinal cortex in the retrieval of spatial memory. hippocampal lesions produced no retrograde amnesia (Ramos 1998, Experiment 2; J.M.J. Ramos, unpubl.). Finally, other data Materials and Methods have suggested that after a group of unoperated animals learn the present reference memory task in the same experimental room, Subjects the animals continue to perform the task well even when a cur- The subjects were 131 male Wistar rats from the breeding colony tain completely conceals the extramaze cues located on the walls of the University of Granada (280–320 g at the time of surgery). near the goal arm (J.M.J. Ramos, unpubl.). Rats were housed singly and maintained on a 12:12-h light/dark In consonance with the results obtained in Experiment 2, cycle. Behavioral testing was performed during the light phase of several studies have shown that the hippocampus is critical for the cycle. All experimental procedures were performed in con- the consolidation and long-term retention of spatial memory formity with European (86/609/EEC) and Spanish (BOE 252, 2005) legislation and were approved by the Ethics Committee for (Ramos 1998, 2000; Riedel et al. 1999; Remondes and Schuman animal research of the University of Granada. 2004; Broadbent et al. 2006; Rossato et al. 2006). These results agree with some theoretical analyses of memory formation that Surgery posit that, following initial learning, a prolonged interaction between the hippocampus and an extensive cortical network of Neurotoxic lesions structures is required to consolidate the information (Alvarez and Under the effects of sodium pentobarbital anesthesia (50 mg/kg Squire 1994; Squire et al. 2001; Texeira et al. 2006). These theo- i.p.; Sigma Chemical), the rats were placed in a David Kopf ste- ries do not assign a specific function to the PRC, although the reotaxic apparatus with the incisor bar adjusted so that lambda close reciprocal connectivity between the hippocampus and the and bregma were level. Rats were randomly assigned to either an PRC and also between the PRC and the neocortex (Scharfman et experimental or a control group. The lesioned subjects received al. 2000) raises the possibility that the perirhinal region contrib- bilateral injections of NMDA (Sigma Chemical, PBS, pH 7.4, 0.07 utes directly and during a long period of time to the activation or M) through the insertion of a 30-gauge stainless steel cannula in retrieval of spatial memories distributed in neocortical regions. eight sites of the perirhinal cortex. The cannula was oriented Therefore, it may be that PRC lesions alter the functional access laterally at 26° from the vertical. The coordinates were derived from the atlas of Paxinos and Watson (1998) and based on the to the neocortical representations of spatial memories, producing anatomical location of the perirhinal cortex, as delineated by a deficit in retrieval. Our data, along with those of previous stud- Burwell and colleagues (Burwell et al. 1995; Burwell and Amaral ies, favor this view. Specifically, Higuchi and Miyashita (1996) 1998). The anteroposterior (AP) stereotaxic coordinates were cal- showed that inferotemporal neurons, a visual memory store- culated relative to bregma, the lateral (L) relative to the midline house in primates, can acquire associative mnemonic codes for and the dorsoventral (V) relative to the top of the skull: ;V = 9.8 ,2.9ע = L ,3.6מ = V = 9.8; AP ,2.4ע = L ,2.5מ = pictures through paired-associative learning. However, lesions of AP V = 9.8. The ,2.8ע = L ,5.8מ = V = 9.8; AP ,3.3ע = L ,4.8מ = the PRC, probably through disruption of backward neural inputs AP to the inferotemporal neurons, destroyed the associational abil- neurotoxin was administered in a 0.5-µL volume at each site ity of the inferotemporal neurons, thus inducing retrograde am- through the cannula that was attached to a 5-µL Hamilton mi- crosyringe. Delivery of the solution was carried out with a Har- nesia. Other studies have suggested that most hippocampal pro- vard Apparatus pump set (model 22) at an infusion rate of 10 jections to the neocortex involve a multisynaptic pathway that µL/h. The cannula was left in situ for an additional 5 min before sequentially progresses through the entorhinal cortex, perirhinal being withdrawn. The rats in the control groups underwent the areas 35 and 36, and then the neocortex (Scharfman et al. 2000). same surgical procedure, except that no solution was adminis- In this regard, Paz et al. (2007) recently demonstrated that the tered through the cannula. medial prefrontal cortex influences hippocampo–cortical interactions. Specifically, using an appetitive trace-conditioning para- Lidocaine lesions digm dependent on the hippocampus, they showed that as learn- In Experiments 2, 3, and 4, all rats were subjected to stereotaxic ing progressed over days, an enhanced transmission from the surgery and implanted bilaterally with 22-gauge stainless steel entorhinal to the perirhinal neurons was observed, suggesting a guide cannulas built in our lab. Each cannula was oriented lat- transfer of hippocampal impulses toward the neocortex (Paz et al. erally at 26° from the vertical and implanted using the following mm from 3.3ע = mm from bregma; L 4.8מ = coordinates: AP 2007). In sum, the above studies, in consonance with the main the midline; V = 5 mm from the skull surface. The cannulas were data of the present research, suggest that the rhinal cortices oc- anchored to the skull by two stainless steel screws and dental cupy a strategic position from which to access neocortical repre- cement. A dummy cannula of 12 mm was inserted into each sentations of recently learned information. cannula guide after surgery to maintain patency. In Experiment The results of this study are also consistent with the obser- 2, an additional group of rats with bilateral guide cannulas im- vation that in amnesic patients more extended and profound planted in the dorsal hippocampus was used. In this group, the www.learnmem.org 594 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Retrograde amnesia after perirhinal cortex lesion

mm from In Experiments 2 and 3, the general procedure used was 3.8מ = following stereotaxic coordinates were used: AP -mm from the midline; V = 2 mm from the identical to that described for the preoperative phase of Experi 2.9ע = bregma; L skull surface. Each rat received the antibiotic Omnamicina 1 mil- ment 1, except that lidocaine or PBS was injected into the PRC or lón (Hoechst Ibérica) for prophylaxis against infections (0.1 mL the dorsal hippocampus daily immediately after training (Experi- intramuscular for 2 d). Rats were given at least 10 d of recovery ment 2) or before a memory test (Experiment 3). In addition, as before beginning training in the radial maze. indicated above, in Experiment 2, in order to accelerate acquisition, an intramaze cue (sandpaper) signaled the location of the Infusion protocol goal arm during learning but not during the memory test. For the 10 d prior to training, a procedure was followed to ac- In Experiment 4, the animals learned an egocentric task in- custom the animals to the infusion process. Once a day, each rat dependent of the hippocampus (Ramos 1998). The general pro- was taken from its cage and restrained gently by the experi- cedure was the same as the one described for the preoperative menter for 3 min. The dummy cannulas were removed during phase of Experiment 1, except that the reward was located in the this time, and the rats could hear the sound of the infusion food cup of the arm situated to the right of the starting arm (for pump. half of the animals) or in the arm situated to the left of the After this habituation procedure, training began and lido- starting arm (the other half of the animals). The arm used for caine hydrochloride at 4% (Sigma; PBS, pH 7.4), which has been starting (N, S, E, or W) was randomized among trials. On the day shown to effectively inactivate the dorsal hippocampus (Broad- after reaching the learning criterion, the animals received lido- bent et al. 2006) and the perirhinal cortex (Winters and Bussey caine or PBS in the PRC 5 min before a test of memory. 2005b), was injected bilaterally in the experimental animals. Control rats received buffer injections. All infusions took place in Histology a room separate from the behavioral testing room. Prior to the infusion, dummy cannulas were removed, and infusion cannulas When the behavioral testing was completed, the rats were deeply measuring 9.7 mm in length from the surface of the skull to the anesthetized with sodium pentobarbital (80 mg/kg i.p.) and per- target region (for the PRC) or 3.4 mm in length (for the dorsal fused intercardially with 0.9% saline, followed by 10% formalin. hippocampus in Experiment 2), were inserted into the guide can- After extraction from the skull, the brains were post-fixed in 10% nulas. Bilateral infusions were made simultaneously, using two formalin for several days and in 10% formalin–30% sucrose until sectioning. Coronal sections (50 µm) were cut on a cryostat מ 5-µL Hamilton microsyringes, 1 min after each training session (Experiment 2) or 5 min before a memory test (Experiments 3 ( 17°C) and stained with cresyl violet, a Nissl stain. and 4). The microsyringes were driven by a Harvard Apparatus pump (model 22), which delivered 1 µL to each hemisphere over Acknowledgments 90 sec. The cannulas were left in situ for an additional 2 min before being withdrawn. This research was supported by a grant from the Spanish Ministry of Education and Science (General Office for Research) and the Apparatus European Regional Development Fund–FEDER–(SEJ2006-03012). I am grateful to Joaquín M.M. Vaquero for his helpful comments A four-arm plus-shaped maze was used. Each arm of the maze on this work. I also thank Juan Carlos Rodríguez and Fernando ן measured 60 cm in length 10 cm in width and was connected Garzón for their valuable technical assistance. to an octagonal central platform 35 cm in diameter. The walls of the central platform were made of transparent Plexiglas and were 15 cm in height. The walls of each arm were made of wood and References measured 5 cm in height. The maze was 60 cm from the floor, Aggleton, J.P., Vann, S.D., Oswald, C.J., and Good, M. 2000. Identifying and a 200 W lightbulb was hanging from the ceiling, 1.2 m above cortical inputs to the rat hippocampus that subserve allocentric the center of the platform. A schematic diagram of the maze and spatial processes: A simple problem with a complex answer. cues in the testing room has been presented elsewhere (Ramos Hippocampus 10: 466–474. 1998, Fig. 1). Aggleton, J.P., Kyd, R.J., and Bilkey, D.K. 2004. When is the perirhinal cortex necessary for the performance of spatial memory tasks? 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During the postoperative relearning in Experiment Dolleman-Van Der Weel, M.J. and Witter, M.P. 1996. Projections from 1, the behavioral procedure followed was identical to that of the the nucleus reuniens thalami to the entorhinal cortex, hippocampal preoperative phase, except that on the first day of relearning no field CA1, and the subiculum in the rat arise from different reward was present. populations of neurons. J. Comp. Neurol. 364: 637–650. www.learnmem.org 595 Learning & Memory Downloaded from learnmem.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

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Perirhinal cortex lesions produce retrograde amnesia for spatial information in rats: Consolidation or retrieval?

Juan M.J. Ramos

Learn. Mem. 2008, 15: Access the most recent version at doi:10.1101/lm.1036308

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